Delivery of RNA to trigger multiple immune pathways

ABSTRACT

RNA encoding an immunogen is co-delivered to non-immune cells as the site of delivery and also to immune cells which infiltrate the site of delivery. The responses of these two cell types to the same delivered RNA lead to two different effects, which interact to produce a strong immune response against the immunogen. The non-immune cells translate the RNA and express the immunogen. Infiltrating immune cells respond to the RNA by expressing type I interferons and pro-inflammatory cytokines which produce a local adjuvant effect which acts on the immunogen-expressing non-immune cells to upregulate major histocompatibility complex expression, thereby increasing presentation of the translated protein to T cells. The effects on the immune and non-immune cells can be achieved by a single delivery of a single RNA e.g., by a single injection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/511,762, filed Oct. 27, 2021, now U.S. Pat. No. 11,596,645, which isa continuation of U.S. application Ser. No. 16/512,541, filed Jul. 16,2019, now U.S. Pat. No. 11,291,682, which is a continuation of U.S.application Ser. No. 15/725,858, filed Oct. 5, 2017, now U.S. Pat. No.10,532,067, which is a divisional of U.S. application Ser. No.13/808,085, filed Mar. 27, 2013, now U.S. Pat. No. 9,801,897, which is a371 National Stage Application of PCT/US2011/043104, filed Jul. 6, 2011,which claims the benefit of U.S. Provisional Application No. 61/361,789,filed Jul. 6, 2010. The complete contents of the above-listedApplications are hereby incorporated herein by reference in theirentireties for all purposes.

TECHNICAL FIELD

This invention is in the field of non-viral delivery of RNA forimmunization.

BACKGROUND ART

The delivery of nucleic acids for immunizing animals has been a goal forseveral years. Various approaches have been tested, including the use ofDNA or RNA, of viral or non-viral delivery vehicles (or even no deliveryvehicle, in a “naked” vaccine), of replicating or non-replicatingvectors, or of viral or non-viral vectors.

There remains a need for further and improved nucleic acid vaccines.

DISCLOSURE OF THE INVENTION

According to the invention, RNA encoding an immunogen is delivered tocells to trigger multiple innate immune response pathways. The deliveredRNA triggers both an endosomal innate immunity receptor (e.g., TLR7) andalso a cytoplasmic innate immunity receptor (e.g., a RNA helicase, suchas MDA5 or RIG-I), thereby enhancing the immune response which iselicited when the RNA-encoded immunogen is expressed.

Thus, the invention provides a method of raising an immune response in avertebrate, comprising administering an immunogen-encoding RNA to thevertebrate such that the RNA: (i) stimulates an endosomal innateimmunity receptor; (ii) stimulates a cytoplasmic innate immunityreceptor; and (iii) is translated to provide expression of theimmunogen.

The invention also provides an immunogen-encoding RNA for use in an invivo method of raising an immune response in a vertebrate, wherein themethod comprises administering the RNA to a vertebrate such that theRNA: (i) stimulates an endosomal innate immunity receptor; (ii)stimulates a cytoplasmic innate immunity receptor; and (iii) istranslated to provide expression of the immunogen.

The invention also provides the use of an immunogen-encoding RNA in themanufacture medicament for raising an in vivo immune response in avertebrate, wherein the RNA is prepared for administration to thevertebrate after which it: (i) stimulates an endosomal innate immunityreceptor; (ii) stimulates a cytoplasmic innate immunity receptor; and(iii) is translated to provide expression of the immunogen.

Administration

The invention involves administration of a RNA molecule to a vertebrate.The administration site will usually be muscle tissue, such as skeletalmuscle. Alternatives to intramuscular administration include, but arenot limited to: intradermal, intranasal, intraocular, subcutaneous,intraperitoneal, intravenous, interstitial, buccal, transdermal, orsublingual administration. Intradermal and intramuscular administrationare two preferred routes.

Administration can be achieved in various ways. For instance, injectionvia a needle (e.g., a hypodermic needle) can be used, particularly forintramuscular, subcutaneous, intraocular, intraperitoneal, orintravenous administration. Needle-free injection can be used as analternative. Intramuscular injection is the preferred way ofadministering RNA according to the invention. Injection into the upperarm, deltoid, or thigh muscle (e.g., anterolateral thigh) is typical.

The administration site includes non-immune cells, such as muscle cells(which may be multinucleated and may be arranged into fascicles) and/orfibroblasts. RNA enters the cytoplasm of these cells after (or while)being administered. Entry can be via endocytosis, e.g., across thesarcolemma of a muscle cell, or across the cell membrane of afibroblast. RNA escapes from the endosomes into the cytoplasm, where itcan be bound by RNA helicases (e.g., in the RIG-I-like receptor familyi.e., RLRs) such as RIG-I (RLR-1), MDA5 (RLR-2) and/or LGP2 (RLR-3).This binding initiates RLR-mediated signaling, thereby triggering afirst innate immune pathway which enhances the immunogenic effect of thedelivered RNA. Even if the delivered RNA is single-stranded, it can formdouble-stranded RNA either during replication or due to its secondarystructure, which means that the RNA can also initiate PKR-mediatedsignaling, again leading to the triggering of a cytoplasmic innateimmune pathway. Both RLR-mediated and PKR-mediated signaling can lead tosecretion of type I interferons (e.g., interferon α and/or β) by thenon-immune cells. The non-immune cells may undergo apoptosis aftertransfection. RLR-mediated signaling in the non-immune cell in thepresence of an expressed immunogen is a potent combination forinitiating an effective immune response.

The administration site also includes immune cells, such as macrophages(e.g., bone marrow derived macrophages), dendritic cells (e.g., bonemarrow derived plasmacytoid dendritic cells and/or bone marrow derivedmyeloid dendritic cells), monocytes (e.g., human peripheral bloodmonocytes), etc. These immune cells can be present at the time ofadministration but will usually infiltrate the site afteradministration. For example, the tissue damage caused by invasiveadministration (e.g., caused by a needle at the administration site) cancause immune cells to infiltrate the damaged area. These infiltratingcells will encounter the RNA, which is now at the delivery site and RNAcan enter the immune cells via endocytosis. Inside the endosomes the RNAcan bind to TLR7 (ssRNA), TLR8 (ssRNA), or TLR3 (dsRNA), therebytriggering a second innate immune pathway. These cells may then secretetype I interferons and/or pro-inflammatory cytokines. The RNA can causethis effect via pattern-recognition receptors, such as toll-likereceptors (e.g., TLR7), intracellular helicases (e.g., RIG-I), and PKR(dsRNA-dependent protein kinase). The RNA may or may not be translatedby the immune cells, and so the immune cells may or may not express theimmunogen. If the immunogen is expressed by the immune cell, then it maybe presented by the immune cell's MHC-I and/or MHC-II. If the immunogenis not expressed by the immune cell, then it may instead be captured bythe immune cell from other cells (e.g., non-immune cells) which hadtaken up RNA and expressed the immunogen, and the immunogen can thus bepresented by the immune cell's MHC-II and/or MHC-I. Antigen presentationwill generally occur in draining lymph nodes after immune cells havemigrated away from the administration site.

Thus, the RNA can separately trigger two innate immune pathways: one viacytoplasmic (e.g., RLR-mediated and/or PKR-mediated) signaling and onevia endosomal (e.g., TLR7-mediated) signaling. These two separatetriggers create an immunostimulatory environment which enhances theimmune response which is elicited when the RNA-encoded immunogen isexpressed as a polypeptide. The two triggers may be provided by the samecell type or by different cell types; e.g., the first trigger could bein a fibroblast whereas the second trigger could be in a plasmacytoiddendritic cell. Where the two triggers are provided by the same celltype, they may even be provided by the same single cell. Usually,however, the two triggers are provided by different cell types. In someembodiments the first trigger (RLR-mediated signaling) occurs inTLR7-negative cells and the second trigger (TLR7-mediated signaling)occurs in RIG-I-negative cells (or, more generally, in RLR-negativecells).

The ability of a RNA to stimulate an endosomal innate immunity receptorsuch as TLR7, or to a cytoplasmic innate immunity receptor such asRIG-I, can be directly detected by known in vitro assays. Indirectdetection of the RNA/receptor interaction can be based on detection ofdownstream events which follow receptor stimulation, such as in vitro orin vivo detection of specific cytokine signatures or gene expressionsignatures associated with particular receptors. It is preferred thatRNA “stimulates” an endosomal innate immunity receptor or a cytoplasmicinnate immunity receptor by binding to that receptor; i.e., the RNA“binds to” the receptor rather than merely “stimulates” it. Assays forbinding of RNAs to these receptors are known in the art.

The RNA can be delivered as naked RNA (e.g., merely as an aqueoussolution of RNA), but to enhance both entry to immune and non-immunecells and also subsequent intercellular effects, the RNA is preferablyadministered in combination with a delivery system, such as aparticulate or emulsion delivery system. Three useful delivery systemsof interest are: (i) liposomes, (ii) non-toxic and biodegradable polymermicroparticles, (iii) cationic submicron oil-in-water emulsions.Liposomes are a preferred delivery system.

Liposomes

Various amphiphilic lipids can form bilayers in an aqueous environmentto encapsulate a RNA-containing aqueous core as a liposome. These lipidscan have an anionic, cationic, or zwitterionic hydrophilic head group.Formation of liposomes from anionic phospholipids dates back to the1960s, and cationic liposome-forming lipids have been studied since the1990s. Some phospholipids are anionic whereas other are zwitterionic andothers are cationic. Suitable classes of phospholipid include, but arenot limited to, phosphatidylethanolamines, phosphatidylcholines,phosphatidylserines, and phosphatidyl-glycerols, and some usefulphospholipids are listed in Table 1. Useful cationic lipids include, butare not limited to, dioleoyl trimethylammonium propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterioniclipids include, but are not limited to, acyl zwitterionic lipids andether zwitterionic lipids. Examples of useful zwitterionic lipids areDPPC, DOPC, and dodecylphosphocholine. The lipids can be saturated orunsaturated. The use of at least one unsaturated lipid for preparingliposomes is preferred. If an unsaturated lipid has two tails, bothtails can be unsaturated, or it can have one saturated tail and oneunsaturated tail.

Liposomes can be formed from a single lipid or from a mixture of lipids.A mixture may comprise: (i) a mixture of anionic lipids, (ii) a mixtureof cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) amixture of anionic lipids and cationic lipids, (v) a mixture of anioniclipids and zwitterionic lipids, (vi) a mixture of zwitterionic lipidsand cationic lipids, or (vii) a mixture of anionic lipids, cationiclipids, and zwitterionic lipids. Similarly, a mixture may comprise bothsaturated and unsaturated lipids. For example, a mixture may compriseDSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/orDMG (anionic, saturated). Where a mixture of lipids is used, not all ofthe component lipids in the mixture need to be amphiphilic; e.g., one ormore amphiphilic lipids can be mixed with cholesterol.

The hydrophilic portion of a lipid can be PEGylated (i.e., modified bycovalent attachment of a polyethylene glycol). This modification canincrease stability and prevent non-specific adsorption of the liposomes.For instance, lipids can be conjugated to PEG using techniques such asthose disclosed in reference 1 and 2. Various lengths of PEG can beused, e.g., between 0.5-8 kDa.

A mixture of DSPC, DlinDMA, PEG-DMG, and cholesterol is used in theexamples.

Liposomes are usually divided into three groups: multilamellar vesicles(MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles(LUV). MLVs have multiple bilayers in each vesicle, forming severalseparate aqueous compartments. SUVs and LUVs have a single bilayerencapsulating an aqueous core; SUVs typically have a diameter <50 nm,and LUVs have a diameter >50 nm. Liposomes useful with and of theinvention are ideally LUVs with a diameter in the range of 50-220 nm.For a composition comprising a population of LUVs with differentdiameters: (i) at least 80% by number should have diameters in the rangeof 20-220 nm, (ii) the average diameter (Zav, by intensity) of thepopulation is ideally in the range of 40-200 nm, and/or (iii) thediameters should have a polydispersity index <0.2. The liposome/RNAcomplexes of reference 37 are expected to have a diameter in the rangeof 600-800 nm and to have a high polydispersity.

Techniques for preparing suitable liposomes are well known in the art;e.g., see references 3 to 5. One useful method is described in reference6 and involves mixing (i) an ethanolic solution of the lipids, (ii) anaqueous solution of the nucleic acid, and (iii) buffer, followed bymixing, equilibration, dilution, and purification. Preferred liposomesof the invention are obtainable by this mixing process.

RNA is preferably encapsulated within the liposomes, and so the liposomeforms a outer layer around an aqueous RNA-containing core. Thisencapsulation has been found to protect RNA from RNase digestion. Theliposomes can include some external RNA (e.g., on the surface of theliposomes), but at least half of the RNA (and ideally all of it) isencapsulated.

Polymeric Microparticles

Various polymers can form microparticles to encapsulate or adsorb RNA.The use of a substantially non-toxic polymer means that a recipient cansafely receive the particles, and the use of a biodegradable polymermeans that the particles can be metabolized after delivery to avoidlong-term persistence. Useful polymers are also sterilizable, to assistin preparing pharmaceutical grade formulations.

Suitable non-toxic and biodegradable polymers include, but are notlimited to, poly(α-hydroxy acids), polyhydroxy butyric acids,polylactones (including polycaprolactones), polydioxanones,polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates,tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones orpolyester-amides, and combinations thereof.

In some embodiments, the microparticles are formed from poly(α-hydroxyacids), such as a poly(lactides) (“PLA”), copolymers of lactide andglycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), andcopolymers of D,L-lactide and caprolactone. Useful PLG polymers includethose having a lactide/glycolide molar ratio ranging, for example, from20:80 to 80:20, e.g., 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, 75:25.Useful PLG polymers include those having a molecular weight between, forexample, 5,000-200,000 Da, e.g., between 10,000-100,000, 20,000-70,000,30,000-40,000, 40,000-50,000 Da.

The microparticles ideally have a diameter in the range of 0.02 μm to 8μm. For a composition comprising a population of microparticles withdifferent diameters at least 80% by number should have diameters in therange of 0.03-7 μm.

Techniques for preparing suitable microparticles are well known in theart; e.g., see references 5, 7 (in particular chapter 7), and 8. Tofacilitate adsorption of RNA, a microparticle may include a cationicsurfactant and/or lipid, e.g., as disclosed in references 9 & 10. Analternative way of making polymeric microparticles is by molding andcuring, e.g., as disclosed in reference 11.

Microparticles of the invention can have a zeta potential of between40-100 mV.

One advantage of microparticles over liposomes is that they are readilylyophilized for stable storage.

RNA can be adsorbed to the microparticles, and adsorption is facilitatedby including cationic materials (e.g., cationic lipids) in themicroparticle.

Oil-In-Water Cationic Emulsions

Oil-in-water emulsions are known for adjuvanting influenza vaccines,e.g., the MF59™ adjuvant in the FLUAD™ product, and the AS03 adjuvant inthe PREPANDRIX™ product. RNA delivery according to the present inventioncan utilize an oil-in-water emulsion, provided that the emulsionincludes one or more cationic molecules. For instance, a cationic lipidcan be included in the emulsion to provide a positive droplet surface towhich negatively-charged RNA can attach.

The emulsion comprises one or more oils. Suitable oil(s) include thosefrom, for example, an animal (such as fish) or a vegetable source. Theoil is ideally biodegradable (metabolizable) and biocompatible. Sourcesfor vegetable oils include nuts, seeds, and grains. Peanut oil, soybeanoil, coconut oil, and olive oil, the most commonly available, exemplifythe nut oils. Jojoba oil can be used, e.g., obtained from the jojobabean. Seed oils include safflower oil, cottonseed oil, sunflower seedoil, sesame seed oil, and the like. In the grain group, corn oil is themost readily available, but the oil of other cereal grains such aswheat, oats, rye, rice, teff, triticale, and the like may also be used.6-10 carbon fatty acid esters of glycerol and 1, 2-propanediol, whilenot occurring naturally in seed oils, may be prepared by hydrolysis,separation and esterification of the appropriate materials starting fromthe nut and seed oils. Fats and oils from mammalian milk aremetabolizable and so may be used. The procedures for separation,purification, saponification, and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil., shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Preferred emulsions comprise squalene, a shark liver oil which is abranched, unsaturated terpenoid(C₃₀H₅₀;[(CH₃)₂C[=CHCH₂CH₂C(CH₃)]₂=CHCH₂]2;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9). Squalene, the saturated analog to squalene, can also beused. Fish oils, including squalene and squalene, are readily availablefrom commercial sources or may be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil phase of an emulsion includes a tocopherol, anyof the α-, β-, γ-, δ-, ε-, or ξ-tocopherols can be used, butα-tocopherols are preferred. D-α-tocopherol and DL-α-tocopherol can bothbe used. A preferred α-tocopherol is DL-α-tocopherol. An oil combinationcomprising squalene and a tocopherol (e.g., DL-α-tocopherol) can beused.

The oil in the emulsion may comprise a combination of oils, e.g.,squalene and at least one further oil. The aqueous component of theemulsion can be plain water (e.g., w.f.i.) or can include furthercomponents, e.g., solutes. For instance, it may include salts to form abuffer, e.g., citrate or phosphate salts, such as sodium salts. Typicalbuffers include: a phosphate buffer; a Tris buffer; a borate buffer; asuccinate buffer; a histidine buffer; or a citrate buffer. A bufferedaqueous phase is preferred, and buffers will typically be included inthe 5-20 mM range.

The emulsion also includes a cationic lipid. Preferably this lipid is asurfactant so that it can facilitate formation and stabilization of theemulsion. Useful cationic lipids generally contain a nitrogen atom thatis positively charged under physiological conditions, e.g., as atertiary or quaternary amine. This nitrogen can be in the hydrophilichead group of an amphiphilic surfactant. Useful cationic lipids include,but are not limited to: 1,2-dioleoyloxy-3-(trimethylammonio) propane(DOTAP), 3′-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecyl-ammonium (DDA, e.g., the bromide),1,2-Dimyristoyl-3-Trimethyl-Ammonium Propane (DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP). Other useful cationiclipids are: benzalkonium chloride (BAK), benzelkonium chloride,cetramide (which contains tetradecyltrimethylammonium bromide andpossibly small amounts of dedecyltrimethylammonium bromide andhexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC),cetyl trimethylammonium chloride (CTAC), N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemethanaminiumchloride (DEBDA), dialkyldimetylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane(acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate, N-alkyl pyridinium salts (e.g., cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C12Me6; C12BU6),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), cationic derivatives ofcholesterol, including but not limited to cholesteryl-3β-oxysuccinamnidoethylenetrimethylarnmonium salt, cholesteryl-3β-oxysuccinamidoethylene-dimethylamine, cholesteryl-3β-carboxyarnidoethylenetrimethylammonium salt, and cholesteryl-3β-carboxyamidoethylenedimethylamine. Other useful cationic lipids aredescribed in refs. 12 & 13.

The cationic lipid is preferably biodegradable (metabolizable) andbiocompatible.

In addition to the oil and cationic lipid, an emulsion can include anon-ionic surfactant and/or a zwitterionic surfactant. Such surfactantsinclude, but are not limited to: the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially polysorbate20 and polysorbate 80; copolymers of ethylene oxide (EO), propyleneoxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which canvary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being ofparticular interest; (octylphenoxy)polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin);polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, andoleyl alcohols (known as Brij surfactants), such as triethyleneglycolmonolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitanesters (commonly known as the Spans), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Preferred surfactants for including in theemulsion are polysorbate 80 (Tween 80; polyoxyethylene sorbitanmonooleate), Span 85 (sorbitan trioleate), lecithin, and Triton X-100.

Mixtures of these surfactants can be included in the emulsion, e.g.,Tween 80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol. Useful mixtures can comprise asurfactant with a HLB value in the range of 10-20 (e.g., polysorbate 80,with a HLB of 15.0) and a surfactant with a HLB value in the range of1-10 (e.g., sorbitan trioleate, with a HLB of 1.8).

Preferred amounts of oil (% by volume) in the final emulsion are between2-20%, e.g., 5-15%, 6-14%, ⁷-13%, ⁸-12%. A squalene content of about4-6% or about 9-11% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion arebetween 0.001% and 8%. For example: polyoxyethylene sorbitan esters(such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%,between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%,between 1.9-2.1%, about 2%, or 0.85-0.95%, or about 1%; sorbitan esters(such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% orabout 1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100)0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers(such as laureth 9) 0.1 to 8%, preferably 0.1% to 10% and in particular0.1 to 1%, or about 0.5%.

The absolute amounts of oil and surfactant, and their ratio, can bevaried within wide limits while still forming an emulsion. A skilledperson can easily vary the relative proportions of the components toobtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 foroil and surfactant is typical (excess oil).

An important parameter for ensuring immunostimulatory activity of anemulsion, particularly in large animals, is the oil droplet size(diameter). The most effective emulsions have a droplet size in thesubmicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the average droplet size is less than 250 nm, e.g.,less than 200 nm, less than 150 nm. The average droplet size is usefullyin the range of 80-180 nm. Ideally, at least 80%, (by number) of theemulsion's oil droplets are less than 250 nm in diameter, and preferablyat least 90%. Apparatuses for determining the average droplet size in anemulsion, and the size distribution, are commercially available. Thesetypically use the techniques of dynamic light scattering and/orsingle-particle optical sensing, e.g., the ACCUSIZER™ and NICOMP™ seriesof instruments available from Particle Sizing Systems (Santa Barbara,USA), or the ZETASIZER™ instruments from Malvern Instruments (UK), orthe Particle Size Distribution Analyzer instruments from Horiba (Kyoto,Japan).

Ideally, the distribution of droplet sizes (by number) has only onemaximum i.e., there is a single population of droplets distributedaround an average (mode), rather than having two maxima. Preferredemulsions have a polydispersity of <0.4, e.g., 0.3, 0.2, or less.

Suitable emulsions with submicron droplets and a narrow sizedistribution can be obtained by the use of microfluidization. Thistechnique reduces average oil droplet size by propelling streams ofinput components through geometrically fixed channels at high pressureand high velocity. These streams contact channel walls, chamber wallsand each other. The results shear, impact, and cavitation forces cause areduction in droplet size. Repeated steps of microfluidization can beperformed until an emulsion, with a desired droplet size average anddistribution are achieved.

As an alternative to microfluidization, thermal methods can be used tocause phase inversion, as disclosed in reference 14. These methods canalso provide a submicron emulsion with a tight particle sizedistribution.

Preferred emulsions can be filter sterilized i.e., their droplets canpass through a 220 nm filter. As well as providing a sterilization, thisprocedure also removes any large droplets in the emulsion.

In certain embodiments, the cationic lipid in the emulsion is DOTAP. Thecationic oil-in-water emulsion may comprise from about 0.5 mg/ml toabout 25 mg/ml DOTAP. For example, the cationic oil-in-water emulsionmay comprise DOTAP at from about 0.5 mg/ml to about 25 mg/mL, from about0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml,from about 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, about 0.5mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3mg/ml., about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12mg/ml, about 18 mg/ml, about 20 mg/ml, about 21.8 mg/ml about 24 mg/ml,etc. In an exemplary embodiment, the cationic oil-in-water emulsioncomprises from about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8mg/ml, 1.2 mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DC Cholesterol. Thecationic oil-in-water emulsion may comprise DC Cholesterol at from about0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationicoil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/mlto about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, fromabout 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml toabout 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, fromabout 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml toabout 4 mg/ml, from about 0. 1 mg/ml to about 3.5 mg/ml, from about 0.1mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, fromabout 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml toabout 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml,about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml,about 4.92 mg/ml, etc. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.62 mg/ml to about 4.92mg/ml DC Cholesterol, such as 2.46 mg/ml.

In certain embodiments, the cationic lipid is DDA. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/mlDDA. For example, the cationic oil-in-water emulsion may comprise DDA atfrom about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml toabout 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, fromabout 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml toabout 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, fromabout 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml toabout 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, fromabout 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml,from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45mg/ml, etc. Alternatively, the cationic oil-in-water emulsion maycomprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about21.6 mg/ml, about 25 mg/ml. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.73 mg/ml to about 1.45mg/ml DDA, such as 1.45 mg/ml.

Certain preferred compositions of the invention for administration to apatient comprise squalene, span 85, polysorbate 80, and DOTAP. Forinstance: squalene may be present at 5-15 mg/ml; span 85 may be presentat 0.5-2 mg/ml; polysorbate 80 may be present at 0.5-2 mg/ml; and DOTAPmay be present at 0.1-10 mg/ml. The emulsion can include the same amount(by volume) of span 85 and polysorbate 80. The emulsion can include moresqualene than surfactant. The emulsion can include more squalene thanDOTAP.

The RNA

The invention involves in vivo delivery of RNA which encodes animmunogen. The RNA triggers two separate innate immunity pathways and isalso translated, leading to expression of the immunogen.

The RNA is +-stranded, and so it can be translated without needing anyintervening replication steps such as reverse transcription.

Preferred +-stranded RNAs are self-replicating. A self-replicating RNAmolecule (replicon) can, when delivered to a vertebrate cell evenwithout any proteins, lead to the production of multiple daughter RNAsby transcription from itself (via an antisense copy which it generatesfrom itself). A self-replicating RNA molecule is thus typically a+-strand molecule which can be directly translated after delivery to acell, and this translation provides a RNA-dependent RNA polymerase whichthen produces both antisense and sense transcripts from the deliveredRNA. Thus, the delivered RNA leads to the production of multipledaughter RNAs. These daughter RNAs, as well as collinear subgenomictranscripts, may be translated themselves to provide in situ expressionof an encoded immunogen, or may be transcribed to provide furthertranscripts with the same sense as the delivered RNA which aretranslated to provide in situ expression of the immunogen. The overallresults of this sequence of transcriptions is a huge amplification inthe number of the introduced replicon RNAs and so the encoded immunogenbecomes a major polypeptide product of the cells.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. These +-stranded replicons are translatedafter delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic −-strand copies of the +-strand delivered RNA. These −-strandtranscripts can themselves be transcribed to give further copies of the+-stranded parent RNA and also to give a subgenomic transcript whichencodes the immunogen. Translation of the subgenomic transcript thusleads to in situ expression of the immunogen by the infected cell.Suitable alphavirus replicons can use a replicase from a Sindbis virus,a Semliki Forest virus, an eastern equine encephalitis virus, aVenezuelan equine encephalitis virus (VEEV), etc. Mutant or wild-typevirus sequences can be used; e.g., the attenuated TC83 mutant of VEEVhas been used in replicons (see reference [15]).

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen. The polymerase canbe an alphavirus replicase, e.g., comprising one or more of alphavirusproteins nsP1, nsP2, nsP3, and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase polyprotein, it is preferredthat a self-replicating RNA molecule of the invention does not encodealphavirus structural proteins. Thus, a preferred self-replicating RNAcan lead to the production of genomic RNA copies of itself in a cell,but not to the production of RNA-containing virions. The inability toproduce these virions means that, unlike a wild-type alphavirus, theself-replicating RNA molecule cannot perpetuate itself in infectiousform. The alphavirus structural proteins which are necessary forperpetuation in wild-type viruses are absent from self-replicating RNAsof the invention and their place is taken by gene(s) encoding theimmunogen of interest, such that the subgenomic transcript encodes theimmunogen rather than the structural alphavirus virion proteins.

Thus, a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an immunogen. Insome embodiments the RNA may have additional (e.g., downstream) openreading frames, e.g., to encode further immunogens (see below) or toencode accessory polypeptides.

A self-replicating RNA molecule can have a 5′ sequence which iscompatible with the encoded replicase.

Self-replicating RNA molecules can have various lengths, but they aretypically 5000-25000 nucleotides long, e.g., 8000-15000 nucleotides, or9000-12000 nucleotides. Thus, the RNA is longer than seen in siRNAdelivery.

A RNA molecule useful with the invention may have a 5′ cap (e.g., a7-methylguanosine). This cap can enhance in vivo translation of the RNA.

The 5′ nucleotide of a RNA molecule useful with the invention may have a5′ triphosphate group. In a capped RNA this may be linked to a7-methylguanosine via a 5′-to-5′ bridge. A 5′ triphosphate can enhanceRIG-I binding.

A RNA molecule may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g., AAUAAA) near its 3′ end.

A RNA molecule useful with the invention will typically besingle-stranded. Single-stranded RNAs can generally initiate an adjuvanteffect by binding to TLR7, TLR8, RNA helicases, and/or PKR. RNAdelivered in double-stranded form (dsRNA) can bind to TLR3, and thisreceptor can also be triggered by dsRNA which is formed either duringreplication of a single-stranded RNA or within the secondary structureof a single-stranded RNA.

A RNA molecule useful with the invention can conveniently be prepared byin vitro transcription (IVT). IVT can use a (cDNA) template created andpropagated in plasmid form in bacteria or created synthetically (forexample by gene synthesis and/or polymerase chain-reaction (PCR)engineering methods). For instance, a DNA-dependent RNA polymerase (suchas the bacteriophage T7, T3 or SP6 RNA polymerases) can be used totranscribe the RNA from a DNA template. Appropriate capping and poly-Aaddition reactions can be used as required (although the replicon'spoly-A is usually encoded within the DNA template). These RNApolymerases can have stringent requirements for the transcribed 5′nucleotide(s) and in some embodiments these requirements must be matchedwith the requirements of the encoded replicase, to ensure that theIVT-transcribed RNA can function efficiently as a substrate for itsself-encoded replicase.

As discussed in reference 16, the self-replicating RNA can include (inaddition to any 5′ cap structure) one or more nucleotides having amodified nucleobase. Thus, the RNA can comprise m5C (5-methylcytidine),m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um(2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine);Am (2′-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A(N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine);io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladcnosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6.-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tin (2′-O-methylinosine); m4C(N4-methycytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosinc); m62Am(N6,N6,0-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine): m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyladenosine);irinomethyluridine; tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); or ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine,7-substituted derivatives thereof, dihydrouracil, pseudouracil,2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil,5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine,5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine,5-fluorocytosine, 5-bromocytosine, N2--dimethylguanine7-deazaguanine,8-azaguanine, 7-deaza-7-substituted guanine,7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine,8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine,2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine,8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine,7-deaza-8-substituted purine, or an abasic nucleotide. For instance, aself-replicating RNA can include one or more modified pyrimidinenucleobases, such as pseudouridine and/or 5-methylcytosine residues. Insome embodiments, however, the RNA includes no modified nucleobases, andmay include no modified nucleotides i.e., all of the nucleotides in theRNA are standard A, C, G and U ribonucleotides (except for any 5′ capstructure, which may include a 7-methylguanosine). In other embodiments,the RNA may include a 5′ cap comprising a 7-methylguanosine, and thefirst 1, 2, or 3 5′ ribonucleotides may be methylated at the 2′ positionof the ribose.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments, it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

Ideally, administered RNA includes fewer than 10 different species ofRNA, e.g., 5, 4, 3, or 2 different species; most preferably, acomposition includes a single RNA species; i.e., all RNA molecules inthe composition (e.g., within a liposome) have the same sequence andsame length.

The Immunogen

RNA molecules used with the invention encode a polypeptide immunogen.After administration of the RNA, the immunogen is translated in vivo andcan elicit an immune response in the recipient. The immunogen may elicitan immune response against a bacterium, a virus, a fungus, or a parasite(or, in some embodiments, against an allergen; and in other embodiments,against a tumor antigen). The immune response may comprise an antibodyresponse (usually including IgG) and/or a cell-mediated immune response.The polypeptide immunogen will typically elicit an immune response whichrecognizes the corresponding bacterial, viral, fungal, or parasite (orallergen or tumor) polypeptide, but in some embodiments the polypeptidemay act as a mimotope to elicit an immune response which recognizes abacterial, viral, fungal, or parasite saccharide. The immunogen willtypically be a surface polypeptide, e.g., an adhesin, a hemagglutinin,an envelope glycoprotein, a spike glycoprotein, etc.

RNA molecules can encode a single polypeptide immunogen or multiplepolypeptides. Multiple immunogens can be presented as a singlepolypeptide immunogen (fusion polypeptide) or as separate polypeptides.If immunogens are expressed as separate polypeptides, then one or moreof these may be provided with an upstream IRES or an additional viralpromoter element. Alternatively, multiple immunogens may be expressedfrom a polyprotein that encodes individual immunogens fused to a shortautocatalytic protease (e.g., foot-and-mouth disease virus 2A protein),or as inteins.

Unlike references 37 and 17, the RNA encodes an immunogen. For theavoidance of doubt, the invention does not encompass RNA which encodes afirefly luciferase or which encodes a fusion protein of E. coliβ-galactosidase or which encodes a green fluorescent protein (GFP).Also, the RNA is not total mouse thymus RNA.

In some embodiments the immunogen elicits an immune response against oneof these bacteria:

-   -   Neisseria meningitidis: useful immunogens include, but are not        limited to, membrane proteins such as adhesins,        autotransporters, toxins, iron acquisition proteins, and factor        H binding protein. A combination of three useful polypeptides is        disclosed in reference 18.    -   Streptococcus pneumoniae: useful polypeptide immunogens are        disclosed in reference 19. These include, but are not limited        to, the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase        precursor (spr0057), spr0096, General stress protein GSP-781        (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and        pneumococcal surface adhesin PsaA.    -   Streptococcus pyogenes: useful immunogens include, but are not        limited to, the polypeptides disclosed in references 20 and 21.    -   Moraxella catarrhalis.    -   Bordetella pertussis: Useful pertussis immunogens include, but        are not limited to, pertussis toxin or toxoid (PT), filamentous        hemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.    -   Staphylococcus aureus: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 22, such as        a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006),        and/or the sta011 lipoprotein.    -   Clostridium tetani: the typical immunogen is tetanus toxoid.    -   Cornynebacterium diphtheriae: the typical immunogen is        diphtheria toxoid.    -   Haemophilus influenzae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in references 23 and 24.    -   Pseudomonas aeruginosa    -   Streptococcus agalactiae: useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 20.    -   Chlamydia trachomatis: Useful immunogens include, but are not        limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12,        OmcA, AtoS, CT547, Eno, HtrA, and MurG (e.g., as disclosed in        reference 25. LcrE (see reference [26]) and HtrA (see reference        [27]) are two preferred immunogens.    -   Chlamydia pneumoniae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 28.    -   Helicobacter pylori: Useful immunogens include, but are not        limited to, CagA, VacA, NAP, and/or urease (see reference [29]).    -   Escherichia coli: Useful immunogens include, but are not limited        to, immunogens derived from enterotoxigenic E. coli (ETEC),        enteroaggregative E. coli (EAggEC), diffusely adhering E. coli        (DAEC), enteropathogenic E. coli (EPEC), extraintestinal        pathogenic E. coli (ExPEC), and/or enterohemorrhagic E. coli        (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and        meningitis/sepsis-associated E. coli (MNEC). Useful UPEC        polypeptide immunogens are disclosed in references 30 and 31.        Useful MNEC immunogens are disclosed in reference 32. A useful        immunogen for several E. coli types is AcfD (see reference        [33]).    -   Bacillus anthracis    -   Yersinia pestis: Useful: immunogens include, but are not limited        to, those disclosed in references 34 and 35.    -   Staphylococcus epidermis    -   Clostridium perfringens or Clostridium botulinums    -   Legionella pneumophila    -   Coxiella burnetti    -   Brucella, such as B.abortus, B.canis, B.melitensis, B.neotomae,        B.ovis, B.suis, B.pinnipediae. Francisella, such as F.novicida,        F.philomimgia, F.tularensis.    -   Neisseria gonorrhoeae    -   Treponema pallidum    -   Haemophilus ducreyi    -   Enterococcus faecalis or Enterococcus faecium    -   Staphylococcus saprophyticus    -   Yersinia enterocolitica    -   Mycobacterium tuberculosis    -   Rickettsia    -   Listeria monocytogenes    -   Vibrio cholerae    -   Salmonella typhi    -   Borrelia burgdorferi    -   Porphyromonas gingivalis    -   Klebsiella

In some embodiments the immunogen elicits an immune response against oneof these viruses:

-   -   Orthomyxovirus: Useful immunogens can be from an influenza A, B        or C virus, such as the hemagglutinin, neuraminidase, or matrix        M2 proteins. Where the immunogen is an influenza A virus        hemagglutinin it may be from any subtype e.g., H1, H2, H3, H4,        H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16.    -   Paramyxoviridae viruses: Viral immunogens include, but are not        limited to, those derived from Pneumoviruses (e.g., respiratory        syncytial virus, RSV), Rubulaviruses (e.g., mumps virus),        Paramyxoviruses (e.g., parainfluenza virus), Metapneumoviruses,        and Morbilliviruses (e.g., measles).    -   Poxviridae: Viral immunogens include, but are not limited to,        those derived from Orthopoxvirus such as Variola vera, including        but not limited to, Variola major and Variola minor.    -   Picornavirus: Viral immunogens include, but are not limited to        those derived from Picornaviruses, such as Enteroviruses,        Rhinoviruses, Heparnaviruses, Cardioviruses, and Aphthoviruses.        In one embodiment, the enterovirus is a poliovirus e.g., a type        1, type 2, and/or type 3 poliovirus. In another embodiment, the        enterovirus is an EV71 enterovirus. In another embodiment, the        enterovirus is a coxsackie A or B virus.    -   Bunyavirus: Viral immunogens include, but are not limited to,        those derived from an Orthobunyavirus, such as California        encephalitis virus, a Phlebovirus, such as Rift Valley Fever        virus, or a Nairovirus, such as Crimean-Congo hemorrhagicfever        virus.    -   Heparnavirus: Viral immunogens include, but are not limited to,        those derived from a Heparnavirus, such as hepatitis A virus        (HAV).    -   Filovirus: Viral immunogens include, but are not limited to,        those derived from a filovirus, such as an Ebola virus        (including a Zaire, Ivory Coast, Reston, or Sudan ebolavirus) or        a Marburg virus.    -   Togavirus: Viral immunogens include, but are not limited to,        those derived from a Togavirus, such as a Rubivirus, an        Alphavirus, or an Arterivirus. This includes rubella virus.    -   Flavivirus: Viral immunogens include, but are not limited to,        those derived from a Flavivirus, such as Tick-borne encephalitis        (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever        virus, Japanese encephalitis virus, Kyasanur Forest Virus, West        Nile encephalitis virus, St. Louis encephalitis virus, Russian        spring-summer encephalitis virus, Powassan encephalitis virus.    -   Pestivirus: Viral immunogens include, but are not limited to,        those derived from a Pestivirus, such as Bovine viral diarrhea        (BVDV), Classical swine fever (CSFV), or Border disease (BDV).    -   Hepadnavirus: Viral immunogens include, but are not limited to,        those derived from a Hepadnavirus, such as Hepatitis B virus. A        composition can include hepatitis B virus surface antigen        (HBsAg).    -   Other hepatitis viruses: A composition can include an immunogen        from a hepatitis C virus, delta hepatitis virus, hepatitis E        virus, or hepatitis G virus.    -   Rhabdovirus: Viral immunogens include, but are not limited to,        those derived from a Rhabdovirus, such as a Lyssavirus (e.g., a        Rabies virus) and Vesiculovirus (VSV).    -   Caliciviridae: Viral immunogens include, but are not limited to,        those derived from Calciviridae, such as Norwalk virus        (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and        Snow Mountain Virus.    -   Coronavirus: Viral immunogens include, but are not limited to,        those derived from a SARS coronavirus, avian infectious        bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine        transmissible gastroenteritis virus (TGEV). The coronavirus        immunogen may be a spike polypeptide.    -   Retrovirus: Viral immunogens include, but are not limited to,        those derived from an Oncovirus, a Lentivirus (e.g., HIV-1 or        HIV-2), or a Spumavirus.    -   Reovirus: Viral immunogens include, but are not limited to,        those derived from an Orthoreovirus, a Rotavirus, an Orbivirus,        or a Coltivirus.    -   Parvovirus: Viral immunogens include, but are not limited to,        those derived from Parvovirus B19.    -   Herpesvirus: Viral immunogens include, but are not limited to,        those derived from a human herpesvirus, such as, by way of        example only, Herpes Simplex Viruses (HSV) (e.g., HSV types 1        and 2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),        Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human        Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).    -   Papovaviruses: Viral immunogens include, but are not limited to,        those derived from Papillomaviruses and Polyomaviruses. The        (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11,        13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, or 65        e.g., from one or more of serotypes 6, 11, 16, and/or 18.    -   Adenovirus: Viral immunogens include those derived from        adenovirus serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of Atlantic salmon), landlocked salmon virus (LSV), Atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

Fungal immunogens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis.Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillusnidulans, Aspergillus terreus. Aspergillus sydowii, Aspergillusflavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candidaalbicans, Candida enolase, Candida tropica/is, Candida glabrata, Candidakrusei, Candida parapsilosis, Candida stellatoidea, Candida kusei,Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candidaguilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomycesdermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasmacapsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp.,Septata intestinalis and Enterocytozoon bieneusi; the less common areBrachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp.,Trachipleistophora spp., Vittaforma spp., Paracoccidioides brasiliensis,Pneumocystis carinii. Pythiumn insidiosum, Pityrosporum ovale,Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beige/i,Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaeaspp., Wangiella spp.,Sporothrix spp., Basidiobolus spp., Conidiobolusspp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp,Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp,Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, andCladosporium spp.

In some embodiments the immunogen elicits an immune response against aparasite from the Plasmodium genus, such as P.falciparum, P.vivax,P.malariae, or P.ovale. Thus, the invention may be used for immunizingagainst malaria. In some embodiments the immunogen elicits an immuneresponse against aparasite from the Caligidae family, particularly thosefrom the Lepeophtheirus and Caligus genera e.g., sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments the immunogen elicits an immune response against:pollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens,e.g., mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g., dog, cat.horse, rat, mouse, etc.); and food allergens (e.g., a gliadin).Important pollen allergens from trees, grasses, and herbs are suchoriginating from the taxonomic orders of Fagales, Oleales, Pinales, andplatanaceae including, but not limited to, birch (Betula), alder(Alnus), hazel (Corylus), hornbeam (Carpinus), and olive (Olea), cedar(Cryptomeria and Juniperus), plane tree (Platanus), the order of Poalesincluding grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis,Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales andUrticales including herbs of the genera Ambrosia, Artemisia, andParietaria. Other important inhalation allergens are those from housedust mites of the genus Dermatophagoides and Euroglyphus, storage mitee.g., Lepidoglyphys, Glycyphagus, and Tyrophagus, those fromcockroaches, midges and fleas e.g., Blatella, Periplancta, Chironomus,and Ctenocephalides, and those from mammals such as cat, dog and horse,venom allergens including such originating from stinging or bitinginsects such as those from the taxonomic order of Hymenoptera includingbees (Apidae), wasps (Vespidea), and ants (Formicoidae).

In some embodiments the immunogen is a tumor antigen selected from: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCPI as well as RAGE,BAGE, GAGE, and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can beused, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors; (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-abl (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, KIA 0205, CDC:-27, and LDLR-FUT; (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung, and ovariancancer), mammaglobin, alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer); (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP 1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma); (c) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer; (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example). In certainembodiments, tumor immunogens include, but are not limited to, p¹5,Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virusantigens, EBNA, human papillomavirus (HPV) antigens, including E6 andE7, hepatitis B and C virus antigens, human T-cell lymphotropic virusantigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA27.29BCAA), CA195, CA 242, CA-50, CAM43, CD68KP1, CO-029, FGF-5, Ga733 (EpCAM),HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90, (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6,TAG72, TLP, TPS, and the like.

Pharmaceutical Compositions

RNA will be administered as a component in a pharmaceutical compositionfor immunizing subjects against various diseases. These compositionswill typically include a pharmaceutically acceptable carrier in additionto the RNA, often as part of a delivery system as described above. Athorough discussion of pharmaceutically acceptable carriers is availablein reference 36.

A pharmaceutical composition of the invention may include one or moresmall molecule immunopotentiators. For example, the composition mayinclude a TLR2 agonist (e.g., Pam3CSK4), a TLR4 agonist (e.g., anaminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist(e.g., imiquimod), a TLR8 agonist (e.g., resiquimod), and/or a TLR9agonist (e.g., IC31). Any such agonist ideally has a molecular weight of<2000 Da. Where a RNA is encapsulated, in some embodiments suchagonist(s) are also encapsulated with the RNA, but in other embodimentsthey are unencapsulated. Where a RNA is adsorbed to a particle, in someembodiments such agonist(s) are also adsorbed with the RNA, but in otherembodiments they are unadsorbed.

Pharmaceutical compositions of the invention may include the particlesin plain water (e.g., w.f.i.) or in a buffer, e.g., a phosphate buffer,a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer,or a citrate buffer. Buffer salts will typically be included in the 5-20mM range.

Pharmaceutical compositions of the invention may have a pH between 5.0and 9.5, e.g., between 6.0 and 8.0.

Compositions of the invention may include sodium salts (e.g., sodiumchloride) to give tonicity. A concentration of 10+2 mg/ml NaCl istypical, e.g., about 9 mg/ml.

Compositions of the invention may include metal ion chelators. These canprolong RNA stability by removing ions which can acceleratephosphodiester hydrolysis. Thus, a composition may include one or moreof EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typicallypresent at between 10-500 μM, e.g., 0.1 mM. A citrate salt, such assodium citrate, can also act as a chelator, while advantageously alsoproviding buffering activity.

Pharmaceutical compositions of the invention may have an osmolality ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g., between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2-phenoxyethanol. Mercury-freecompositions are preferred, and preservative-free vaccines can beprepared.

Pharmaceutical compositions of the invention are preferably sterile.

Pharmaceutical compositions of the invention are preferablynon-pyrogenic, e.g., containing <1 EU (endotoxin unit, a standardmeasure) per dose, and preferably <0.1 EU per dose.

Pharmaceutical compositions of the invention are preferably gluten free.

Pharmaceutical compositions of the invention may be prepared in unitdose form. In some embodiments a unit dose may have a volume of between0.1-1.0 ml, e.g., about 0.5 ml.

The compositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration, e.g., by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration, e.g., asspray or drops. Injectables for intramuscular administration aretypical.

Compositions comprise an immunologically effective amount of RNA, aswell as any other components, as needed. By ‘immunologically effectiveamount’, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group of the individual to be treated (e.g., non-humanprimate, primate, etc.), the capacity of the individual's immune systemto synthesize antibodies, the degree of protection desired, theformulation of the vaccine, the treating doctor's assessment of themedical situation, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials. The RNA content of compositions of the inventionwill generally be expressed in terms of the amount of RNA per dose. Apreferred dose has ≤10 μg RNA, and expression can be seen at much lowerlevels, e.g., <1 μg/dose, <100ng/dose, <10ng/dose, <1ng/dose, etc.

The invention also provides a delivery device (e.g., syringe, nebulizer,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a vertebrate subject.

RNAs are not delivered in combination with ribosomes and sopharmaceutical compositions of the invention are ribosome-free.

Methods of Treatment and Medical Uses

RNA delivery according to the invention is for eliciting an immuneresponse in vivo against an immunogen of interest. The immune responseis preferably protective and preferably involves antibodies and/orcell-mediated immunity. The method may raise a booster response.

By raising an immune response the vertebrate can be protected againstvarious diseases and/or infections, e.g., against bacterial and/or viraldiseases as discussed above. RNA-containing compositions areimmunogenic, and are more preferably vaccine compositions. Vaccinesaccording to the invention may either be prophylactic (i.e., to preventinfection) or therapeutic (i.e., to treat infection), but will typicallybe prophylactic.

The vertebrate is preferably a mammal, such as a human or a largeveterinary mammal (e.g., horses, cattle, deer, goats, pigs). Where thevaccine is for prophylactic use, the human is preferably a child (e.g.,a toddler or infant) or a teenager; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus, a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g., >50 years old, >60 years old, and preferably >65years), the young (e.g., <5 years old), hospitalized patients,healthcare workers, armed service and military personnel, pregnantwomen, the chronically ill, or immunodeficient patients. The vaccinesare not suitable solely for these groups, however, and may be used moregenerally in a population.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g., subcutaneously, intraperitoneally, intravenously,intramuscularly, or to the interstitial space of a tissue; unlikereference 37, intraglossal injection is not typically used with thepresent invention), or mucosally, such as by rectal, oral (e.g., tablet,spray), vaginal, topical, transdermal or transcutaneous, intranasal,ocular, aural, pulmonary, or other mucosal administration. Injection maybe via a needle (e.g., a hypodermic needle), but needle-free injectionmay alternatively be used. A typical intramuscular dose is 0.5 mL

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10, weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment multiple doses may be administered approximately 6 weeks, 10weeks, and 14 weeks after birth, e.g., at an age of 6 weeks, 10 weeks,and 14 weeks, as often used in the World Health Organization's ExpandedProgram on Immunization (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g., about 7, 8,or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g., about 6, 8, 10, or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g., about 7, 8,or 9 weeks apart followed by one or more booster doses about 6 months to1 year after the third primary dose, e.g., about 6, 8, 10, or 12 monthsafter the third primary dose.

General Embodiments

In some embodiments of the invention, the RNA includes no modifiednucleotides (see above). In other embodiments the RNA can optionallyinclude at least one modified nucleotide, provided that one or more ofthe following features (already disclosed above) is also required:

-   -   A. Where the RNA is delivered with a liposome, the liposome        comprises DSDMA, DODMA, DLinDMA, and/or DLenDMA.    -   B. Where the RNA is encapsulated in a liposome, the hydrophilic        portion of a lipid in the liposome is PEGylated.    -   C. Where the RNA is encapsulated in a liposome, at least 80% by        number of the liposomes have diameters in the range of 20-220        nm.    -   D. Where the RNA is delivered with a microparticle, the        microparticle is a non-toxic and biodegradable polymer        microparticle.    -   E. Where the RNA is delivered with a microparticle, the        microparticles have a diameter in the range of 0.02 μm to 8 μm.    -   F. Where the RNA is delivered with a microparticle, at least 80%        by number of the microparticles have a diameter in the range of        0.03-7 μm.    -   G. Where the RNA is delivered with a microparticle, the        composition is lyophilized.    -   H. Where the RNA is delivered with an emulsion, the emulsion        comprises a biodegradable oil (e.g., squalene).    -   I. Where the RNA is delivered with an emulsion, the emulsion        includes one or more cationic molecules e.g., one or more        cationic lipids.    -   J. The RNA has a 3′ poly-A tail, and the immunogen can elicit an        immune response in vivo against a bacterium, a virus, a fungus,        or a parasite.    -   K. The RNA is delivered in combination with a metal ion chelator        with a delivery system selected from: (i) liposomes, (ii)        non-toxic and biodegradable polymer microparticles, (iii)        cationic submicron oil-in-water emulsions.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology, and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references38-44, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g., a composition “comprising” X may consist exclusively of X or mayinclude something additional, e.g., X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x 10%.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to charge, to cations, to anions, to zwitterions, etc., aretaken at pH 7.

TLR3 is the Toll-like receptor 3. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR3agonists include poly(I:C). “TLR3” is the approved HGNC name for thegene encoding this receptor, and its unique HGNC ID is HGNC:11849. TheRefSeq sequence for the human TLR3 gene is GI:2459625.

TLR7 is the Toll-like receptor 7. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR7agonists include e.g., imiquimod. “TLR7” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15631.The RefSeq sequence for the human TLR7 gene is GI:67944638.

TLR8 is the Toll-like receptor 8. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR8agonists include e.g., resiquimod. “TLR8” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15632.The RefSeq sequence for the human TLR8 gene is GI:20302165.

The RIG-I-like receptor (“RLR”) family includes various RNA helicaseswhich play key roles in the innate immune system [45]. RLR-1 (also knownas RIG-I or retinoic acid inducible gene I) has two caspase recruitmentdomains near its N-terminus. The approved HGNC name for the geneencoding the RLR-1 helicase is “DDX58” (for DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 58) and the unique HGNC ID is HGNC:19102. The RefSeqsequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known asMDA5 or melanoma differentiation-associated gene 5) also has two caspaserecruitment domains near its N-terminus. The approved HGNC name for thegene encoding the RLR-2 helicase is “IFIH1” (for interferon induced withhelicase C domain 1) and the unique HGNC ID is HGNC:18873. The RefSeqsequence for the human RLR-2 gene is GI: 27886567. RLR-3 (also known asLGP2 or laboratory of genetics and physiology 2) has no caspaserecruitment domains. The approved HGNC name for the gene encoding theRLR-3 helicase is “DHX58” (for DEXH (Asp-Glu-X-His) box polypeptide 58)and the unique HGNC ID is HGNC:29517. The RefSeq sequence for the humanRLR-3 gene is GI: 149408121.

PKR is a double-stranded RNA-dependent protein kinase. It plays a keyrole in the innate immune system. “EIF2AK2” (for eukaryotic translationinitiation factor 2-alpha kinase 2) is the approved HGNC name for thegene encoding this enzyme, and its unique HGNC TD is HGNC:9437. TheRefSeq sequence for the human PKR gene is GI:208431825.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a gel with stained RNA. Lanes show: (1) markers, (2) nakedreplicon, (3) replicon after RNase treatment, (4) replicon encapsulatedin liposome, (5) liposome after RNase treatment, (6) liposome treatedwith RNase then subjected to phenol/chloroform extraction.

FIG. 2 is an electron micrograph of liposomes.

FIG. 3 shows protein expression (as relative light units, RLU) at days1, 3 and 6 after delivery of RNA as a virion-package replicon (squares),naked RNA (triangles), or as microparticles (circles).

FIG. 4 shows a gel with stained RNA. Lanes show: (1) markers, (2) nakedreplicon, (3) replicon encapsulated in liposome, (4) liposome treatedwith RNase then subjected to phenol/chloroform extraction.

FIG. 5 shows protein expression at days 1, 3, and 6 after delivery ofRNA as a virion-packaged replicon (squares), as naked RNA (diamonds), orin liposomes (+=0.1μg, x=1 μg).

FIG. 6 shows protein expression at days 1, 3, and 6 after delivery offour different doses of liposome-encapsulated RNA.

FIG. 7 shows anti-F IgG titers in animals receiving virion-packagedreplicon (VRP or VSRP), 1 μg naked RNA, and 1p g liposome-encapsulatedRNA.

FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA,and 0.1 μg or 1 μg liposome-encapsulated RNA.

FIG. 9 shows neutralizing antibody titers in animals receiving VRP oreither 0.1 μg or 1 μg liposome-encapsulated RNA.

FIG. 10 shows expression levels after delivery of a replicon as nakedRNA (circles), liposome-encapsulated RNA (triangle & square), or as alipoplex (inverted triangle).

FIG. 11 shows F-specific IgG titers (2 weeks after second dose) afterdelivery of a replicon as naked RNA (0.01-1jpg), liposome-encapsulatedRNA (0.01-10jig), or packaged as a virion (VRP, 10⁶ infectious units orIU).

FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares)after delivery of a replicon as naked RNA (1 μg), liposome-encapsulatedRNA (0.1 or 1 μg), or packaged as a virion (VRP, 10⁶ IU). Titers innaive mice are also shown. Solid lines show geometric means.

FIG. 13 shows intracellular cytokine production after restimulation withsynthetic peptides representing the major epitopes in the F protein, 4weeks after a second dose. The y-axis shows the % cytokine+ of CD8+CD4-.

FIGS. 14A and 14B show F-specific IgG titers (mean log₁₀ titers+std dev)over 63 days (FIG. 14A) and 210 days (FIG. 14B) after immunization ofcalves. The four lines are easily distinguished at day 63 and are, frombottom to top: PBS negative control; liposome-delivered RNA;emulsion-delivered RNA; and the “Triangle 4” product.

FIGS. 15A and 15B show IFN-P (FIG. 15A) and IL-6 (FIG. 15B) released byfibroblasts. The graphs include two sets of 4 bars. The left quartet arefor control mice; the right quartet are for RNA-immunized mice. The 4bars in each quartet, from left to right, show data from rig-i +/−,rig-i −/−, mda5+/− and mda5 −/− mice. Figures are μg/ml.

FIGS. 16A and 16B show IL-6 (FIG. 16A) and IFNα (FIG. 16B) (μg/ml)released by pDC. There are 4 pairs of bars, from left to right: control;immunized with RNA+DOTAP; immunized with RNA+lipofectamine; andimmunized with RNA in liposomes. In each pair the black bar is wild-typemice, grey is rsql mutant.

MODES FOR CARRYING OUT THE INVENTION

RNA Replicons

Various replicons are used below. In general these are based on a hybridalphavirus genome with non-structural proteins from Venezuelan equineencephalitis virus (VEEV), a packaging signal from Sindbis virus, and a3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10kblong and has a poly-A tail.

Plasmid DNA encoding alphavirus replicons (named: pT7-mvEEV-FL.RSVF orA317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a templatefor synthesis of RNA in vitro. The replicons contain the alphavirusgenetic elements required for RNA replication but lack those encodinggene products necessary for particle assembly; the structural proteinsare instead replaced by a protein of interest (either a reporter, suchas SEAP or GFP, or an immunogen, such as full-length RSV F protein) andso the replicons are incapable of inducing the generation of infectiousparticles. A bacteriophage (T7 or SP6) promoter upstream of thealphavirus cDNA facilitates the synthesis of the replicon RNA in vitroand a hepatitis delta virus (HDV) ribozyme immediately downstream of thepoly(A)-tail generates the correct 3-end through its self-cleavingactivity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP andUTP) following the instructions provided by the manufacturer (Ambion).Following transcription, the template DNA was digested with TURBO™ DNase(Ambion). The replicon RNA was precipitated with LiCi and reconstitutedin nuclease-free water. Uncapped RNA was capped post-transcriptionallywith Vaccinia Capping Enzyme (VCE) using the SCRIPTCAP™ m7G CappingSystem (Epicentre Biotechnologies) as outlined in the user manual;replicons capped in this way are given the “v” prefix e.g., vA317 is theA317 replicon capped by VCE. Post-transcriptionally capped RNA wasprecipitated with LiCi and reconstituted in nuclease-free water. Theconcentration of the RNA samples was determined by measuring OD_(260nm).Integrity of the in vitro transcripts was confirmed by denaturingagarose gel electrophoresis.

PLG Adsorption

Microparticles were made using 500 mg of PLG RG503 (50:50lactide/glycolide molar ratio, MW˜30 kDa) and 20 mg DOTAP using an OmniMacro Homogenizer. The particle suspension was shaken at 150 rpmovernight and then filtered through a 40 μm sterile filter for storageat 2-8° C. Self-replicating RNA was adsorbed to the particles. Toprepare 1 mL of PLG/RNA suspension the required volume of PLG particlesuspension was added to a vial and nuclease-free water was added tobring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise tothe PLG suspension, with constant shaking. PLG/RNA was incubated at roomtemperature for 30 min. For 1 mL of reconstituted suspension, 45 mgmannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials werefrozen at −80° C. and lyophilized.

To evaluate RNA adsorption, 100 μL particle suspension was centrifugedat 10,000 rpm for 5 min and supernatant was collected. PLG/RNA wasreconstituted using 1 mL nuclease-free water. To 100 μL particlesuspension (1p g RNA), 1 mg heparin sulfate was added. The mixture wasvortexed and allowed to sit at room temperature for 30 min for RNAdesorption. Particle suspension was centrifuged and supernatant wascollected.

For RNAse stability, 100 μL particle suspension was incubated with6.4mAU of RNAse A at room temperature for 30 min. RNAse was inactivatedwith 0.126mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparinsulfate was added to desorb the RNA followed by centrifugation. Thesupernatant samples containing RNA were mixed with formaldehyde loaddye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel(460ng RNA loaded per lane).

To assess expression, Balb/c mice were immunized with 1 μg RNA in 100 μLintramuscular injection volume (50 μL/leg) on day 0. Sera were collectedon days 1, 3, and 6. Protein expression was determined using achemiluminescence assay. As shown in FIG. 3 , expression was higher whenRNA was delivered by PLG (triangles) than without any delivery particle(circles).

Cationic Nanoemulsion

An oil-in-water emulsion was prepared by microfluidizing squalene, span85, polysorbate 80, and varying amounts of DOTAP. Briefly, oil solublecomponents (squalene, span 85, cationic lipids, lipid surfactants) werecombined in a beaker, lipid components were dissolved in organicsolvent. The resulting lipid solution was added directly to the oilphase. The solvent was allowed to evaporate at room temperature for 2hours in a fume hood prior to combining the aqueous phase andhomogenizing the sample to provide a homogeneous feedstock. The primaryemulsions were passed three to five times through a microfluidizer withan ice bath cooling coil. The batch samples were removed from the unitand stored at 4° C.

This emulsion is thus similar to the commercial MF59T′ adjuvant, butsupplemented by a cationic DOTAP to provide a cationic nanoemulsion(“CNE”). The final composition of emulsion “CNE17” was squalene (4.3% byweight), span 85 (0.5% by weight), polysorbate 80 (0.5% by weight),DOTAP (1.4 mg/ml), in 10 mM citrate buffer, pH 6.5.

RNA adsorbs to the surface of the oil droplets in these cationicemulsions. To adsorb RNA, a RNA solution is diluted to the appropriateconcentration in RNAse free water and then added directly into an equalvolume of emulsion while vortexing lightly. The solution is allowed tosit at room temperature for approximately 2 hours to allow adsorption.The resulting solution is diluted to the required RNA concentrationprior to administration.

Liposomal Encapsulation

RNA was encapsulated in liposomes made by the method of references 6 and46. The liposomes were made of 10%, DSPC (zwitterionic), 40% DlinDMA(cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). Theseproportions refer to the % moles in the total liposome. DlinDMA(1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized usingthe procedure of reference 1. DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme.Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)], ammonium salt), DOTAP(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol(3p-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)were from Avanti Polar Lipids.

Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon wasdissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these weremixed with 2 ml of buffer followed by 1 hour of equilibration. Themixture was diluted with 6 ml buffer then filtered. The resultingproduct contained liposomes, with ˜ 95% encapsulation efficiency.

For example, in one particular method, fresh lipid stock solutions wereprepared in ethanol. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg ofcholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mLof ethanol. The freshly prepared lipid stock solution was gently rockedat 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL ofthe stock was added to 1.245 mL ethanol to make a working lipid stocksolution of 2 mL. This amount of lipids was used to form liposomes with250 μg RNA. A 2 mL working solution of RNA was also prepared from astock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mLglass vials (with stir bars) were rinsed with RNAse Away solution(Molecular BioProducts) and washed with plenty of MilliQ water beforeuse to decontaminate the vials of RNAses. One of the vials was used forthe RNA working solution and the others for collecting the lipid and RNAmixes (as described later). The working lipid and RNA solutions wereheated at 37° C. for 10 min before being loaded into 3cc LUER-LOK©syringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe.Syringes containing RNA and the lipids were connected to a T mixer(PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing(fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internaldiameter and a 3 mm outer diameter; obtained from Idex Health Science).The outlet from the T mixer was also FEP tubing. The third syringecontaining the citrate buffer was connected to a separate piece oftubing.

All syringes were then driven at a flow rate of 7 mL/min using a syringepump. The tube outlets were positioned to collect the mixtures in a 20mL glass vial (while stirring). The stir bar was taken out and theethanol/aqueous solution was allowed to equilibrate to room temperaturefor 1 h. 4 ml of the mixture was loaded into a 5 cc syringe, which wasconnected to a piece of FEP tubing and in another 5 cc syringe connectedto an equal length of FEP tubing, an equal amount of 100 mM citratebuffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flowrate using the syringe pump and the final mixture collected in a 20 mLglass vial (while stirring). Next, the mixture collected from the secondmixing step (liposomes) were passed through a Mustang Q membrane (ananion-exchange support that binds and removes anionic molecules,obtained from Pall Corporation). Before using this membrane for theliposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mMcitrate buffer (pH 6) were successively passed through it. Liposomeswere warmed for 10 min at 37° C. before passing through the membrane.Next, liposomes were concentrated to 2 mL and dialyzed against 10-15volumes of 1 X PBS using by tangential flow filtration before recoveringthe final product. The TFF system and hollow fiber filtration membraneswere purchased from Spectrum Labs (Rancho Dominguez) and were usedaccording to the manufacturer's guidelines. Polysulfone hollow fiberfiltration membranes with a 100 kDa pore size cutoff and 8 cm² surfacearea were used. For in vitro and in vivo experiments formulations werediluted to the required RNA concentration with 1×PBS.

FIG. 2 shows an example electron micrograph of liposomes prepared bythese methods. These liposomes contain encapsulated RNA encodingfull-length RSV F antigen. Dynamic light scattering of one batch showedan average diameter of 141 nm (by intensity) or 78 nm (by number).

The percentage of encapsulated RNA and RNA concentration were determinedby Quant-iT RiboGreen RNA reagent kit (Invitrogen), followingmanufacturer's instructions. The ribosomal RNA standard provided in thekit was used to generate a standard curve. Liposomes were diluted 10× or100× in 1X TE buffer (from kit) before addition of the dye. Separately,liposomes were diluted 10× or 100× in 1X TE buffer containing 0.5%Triton X before addition of the dye (to disrupt the liposomes and thusto assay total RNA). Thereafter an equal amount of dye was added to eachsolution and then ˜180 μL of each solution after dye addition was loadedin duplicate into a 96 well tissue culture plate. The fluorescence (Ex485 nm, Em 528 nm) was read on a microplate reader. All liposomeformulations were dosed in vivo based on the encapsulated amount of RNA.

Encapsulation in liposomes was shown to protect RNA from RNasedigestion. Experiments used 3.8 mAU of RNAse A per microgram of RNA,incubated for 30 minutes at room temperature. RNAse was inactivated withProteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added toextract the RNA from the lipids into the aqueous phase. Samples weremixed by vortexing for a few seconds and then placed on a centrifuge for15 minutes at 12k RPM. The aqueous phase (containing the RNA) wasremoved and used to analyze the RNA. Prior to loading (400 ng RNA perwell) all the samples were incubated with formaldehyde loading dye,denatured for 10 minutes at 65° C. and cooled to room temperature.Ambion MILLENNIUM™ markers were used to approximate the molecular weightof the RNA construct. The gel was run at 90 V. The gel was stained using0.1% SYBR gold according to the manufacturer's guidelines in water byrocking at room temperature for 1 hour. FIG. 1 shows that RNasecompletely digests RNA in the absence of encapsulation (lane 3). RNA isundetectable after encapsulation (lane 4), and no change is seen ifthese liposomes are treated with RNase (lane 4). After RNase-treatedliposomes are subjected to phenol extraction, undigested RNA is seen(lane 6). Even after 1 week at 4° C. the RNA could be seen without anyfragmentation (FIG. 4 , arrow). Protein expression in vivo was unchangedafter 6 weeks at 4° C. and one freeze-thaw cycle. Thus,liposome-encapsulated RNA is stable.

To assess in vivo expression of the RNA a reporter enzyme (SEAP;secreted alkaline phosphatase) was encoded in the replicon, rather thanan immunogen. Expression levels were measured in sera diluted 1:4 in 1XPhospha-Light dilution buffer using a chemiluminescent alkalinephosphate substrate. 8-10 week old BALB/c mice (5/group) were injectedintramuscularly on day 0, 50 μL per leg with 0.1 μg or 1 μg RNA dose.The same vector was also administered without the liposomes (in RNasefree 1X PBS) at 1 μg. Virion-packaged replicons were also tested.Virion-packaged replicons used herein (referred to as “VRPs”) wereobtained by the methods of reference 47, where the alphavirus repliconis derived from the mutant VEEV or a chimera derived from the genome ofVEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbisvirus packaging signal (PS), packaged by co-electroporating them intoBHK cells with defective helper RNAs encoding the Sindbis virus capsidand glycoprotein genes.

As shown in FIG. 5 , encapsulation increased SEAP levels by about 1%₂log at the 1p g dose, and at day 6 expression from a 0.1 μg encapsulateddose matched levels seen, with 1p g unencapsulated dose. By day 3expression levels exceeded those achieved with VRPs (squares). Thus,expression increased when the RNA was formulated in the liposomesrelative to the naked RNA control, even at a 10× lower dose. Expressionwas also higher relative to the VRP control, but the kinetics ofexpression were very different (see FIG. 5 ). Delivery of the RNA withelectroporation resulted in increased expression relative to the nakedRNA control, but these levels were lower than with liposomes.

To assess whether the effect seen in the liposome groups was due merelyto the liposome components, or was linked to the encapsulation, thereplicon was administered in encapsulated form (with two differentpurification protocols, 0.1 μg RNA), or mixed with the liposomes aftertheir formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as nakedRNA (1p g). FIG. 10 shows that the lipoplex gave the lowest levels ofexpression, showing that shows encapsulation is essential for potentexpression.

Further SEAP experiments showed a clear dose response in vivo, withexpression seen after delivery of as little as 1 ng RNA (FIG. 6 ).Further experiments comparing expression from encapsulated and nakedreplicons indicated that 0.01p g encapsulated RNA was equivalent to 1sgof naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-foldhigher at day 6.

Rather than looking at average levels in the group, individual animalswere also studied. Whereas several animals were non-responders to nakedreplicon, encapsulation eliminated non-responders.

Further experiments replaced DlinDMA with DOTAP. Although the DOTAPliposomes gave better expression than naked replicon, they were inferiorto the DlinDMA liposomes (2- to 3-fold difference at day 1).

To assess in vivo immunogenicity a replicon was constructed to expressfull-length F protein from respiratory syncytial virus (RSV). This wasdelivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), orpackaged in virions (10⁶ IU; “VRP”) at days 0 and 21. FIG. 7 showsanti-F IgG titers 2 weeks after the second dose, and the liposomesclearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, bywhich point there was no statistical difference between the encapsulatedRNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group.Neutralization titers (measured as 60% plaque reduction, “PRNT60”) werenot significantly different in these three groups 2 weeks after thesecond dose (FIG. 9 ). FIG. 12 shows both IgG and PRNT titers 4 weeksafter the second dose.

FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

Further experiments compared F-specific IgG titers in mice receivingVRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulatedRNA. Titer ratios (VRP:liposome) at various times after the second dosewere as follows:

2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1   l μg 2.3 0.9 0.9

Thus, the liposome-encapsulated RNA induces essentially the samemagnitude of immune response as seen with virion delivery.

Further experiments showed superior F-specific IgG responses with a 10μg dose, equivalent responses for 1p g and 0.1p g doses, and a lowerresponse with a 0.01p g dose. FIG. 11 shows IgG titers in mice receivingthe replicon in naked form at 3 different doses, in liposomes at 4different doses, or as VRP (106 IU). The response seen with 1 μgliposome-encapsulated RNA was statistically insignificant (ANOVA) whencompared to VRP, but the higher response seen with 10 μgliposome-encapsulated RNA was statistically significant (p<0.05) whencompared to both of these groups.

A further study confirmed that the 0.1p g of liposome-encapsulated RNAgave much higher anti-F IgG responses (15 days post-second dose) than0.1 μg of delivered DNA, and even was more immunogenic than 20 μgplasmid DNA encoding the F antigen, delivered by electroporation (Elgen™DNA Delivery System, Inovio).

A further study was performed in cotton rats (Sigmodon hispidis) insteadof mice. At a 1p g dose liposome encapsulation increased F-specific IgGtiters by 8.3-fold compared to naked RNA and increased PRNT titers by9.5-fold. The magnitude of the antibody response was equivalent to thatinduced by 5×10⁶ IU VRP. Both naked and liposome-encapsulated RNA wereable to protect the cotton rats from RSV challenge (1×10⁵ plaque formingunits), reducing lung viral load by at least 3.5 logs. Encapsulationincreased the reduction by about 2-fold.

A large-animal study was performed in cattle. Cows were immunized with66 μg of replicon encoding full-length RSV F protein at days 0, 21, 86,& 146, formulated either inside liposomes or with the CNE17 emulsion.PBS alone was used as a negative control, and a licensed vaccine wasused as a positive control (“Triangle 4′” from Fort Dodge, containingkilled virus). FIGS. 14A and 14B show F-specific IgG titers over thefirst 63 days. The RNA replicon was immunogenic in the cows using bothdelivery systems, although it gave lower titers than the licensedvaccine. All vaccinated cows showed F-specific antibodies after thesecond dose, and titers were very stable from the period of 2 to 6 weeksafter the second dose (and were particularly stable for the RNAvaccines). The titers with the liposome delivery system were moretightly clustered than with the emulsion.

The data from this study provide proof of concept for RNA replicon RSVvaccines in large animals, with two of the five calves in theemulsion-adjuvanted group demonstrating good neutralizing antibodytiters after the third vaccination, as measured by thecomplement-independent HRSV neutralization assay. In acomplement-enhanced HRSV neutralization assay all vaccinated calves hadgood neutralizing antibody titers after the second RNA vaccinationregardless of the formulation. Furthermore, both RNA vaccines elicitedF-specific serum IgG titers that were detected in a few calves after thesecond vaccination and in all calves after the third vaccination.MF59T′-adjuvanted RSV-F was able to boost the IgG response in allpreviously vaccinated calves, and to boost complement-independent HRSVneutralization titers of calves previously vaccinated with RNA.

Mechanism of Action

Bone marrow derived dendritic cells (pDC) were obtained from wild-typemice or the “Resq” (rsq]) mutant strain. The mutant strain has a pointmutation at the amino terminus of its TLR7 receptor which abolishes TLR7signaling without affecting ligand binding (see reference [48]). Thecells were stimulated with replicon RNA formulated with DOTAP,lipofectamine 2000, or inside a liposome. As shown in FIGS. 16A and 16B,IL-6 (FIG. 16A) and INFα (FIG. 16B) were induced in WT cells, but thisresponse was almost completely abrogated in mutant mice. These resultsshow that TLR7 is required for RNA recognition in immune cells, and thatliposome-encapsulated replicons can cause immune cells to secrete highlevels of both interferons and pro-inflammatory cytokines.

The involvement of TLR7 was further investigated by comparing responsesin wild type (WT) C57BL/6 mice and in the “Resq” mutant strain. Mice (5per group) were given bilateral intramuscular vaccinations (50 μL perleg) on days 0 and 21 with 1 μg self-replicating RNA (“vA317”, encodingthe surface fusion glycoprotein of RSV) formulated in liposomes (40%DlinDMA, 10% DSPC, 48% cholesterol, 2% PEG-DMG conjugate), or with 2 μgof RSV-F protein adjuvanted with aluminum hydroxide.

Serum was collected for immunological analysis on days 14 (2wp1) and 35(2wp2). F-specific serum IgG titers (GMT) were as follows:

RNA vaccine Protein vaccine Day WT Resq WT Resq Total IgG 14 1038 1452324 2601 35 9038 1224 27211 17150 IgG 1 14 25 25 3657 2974 35 125 12534494 26459 IgG 2c 14 1941 211 25 25 35 35804 2080 125 125

With the protein vaccine, F-specific serum IgG titers were comparablebetween the wild type and Resq C56BL/6 mice i.e., immunogenicity of theprotein vaccine was not dependent on TLR7. In contrast, theself-replicating RNA formulated in liposomes showed a 7-fold decrease inF-specific serum IgG titers after both vaccinations, indicating at leasta partial dependence on TLR7 for the immunogenicity of the RNA vaccine.

The results also show that the RNA vaccine can elicit primarily aT_(h)l-type immune response.

Further experiments were performed with the same RNA and the same mutantmice. Mice were given bilateral intramuscular vaccinations (50 μL perleg) on days 0 and 21 with 1p g of the RNA replicon, formulated eitherwith a submicron cationic oil-in-water nanoemulsion (squalene, span 85,polysorbate 80, DOTAP) or with liposomes (40% DlinDMA, 10% DSPC, 48%cholesterol, 2% PEG-conjugated DMG). For comparison, 2 μg ofalum-adjuvanted F protein was used. Sera were collected forimmunological analysis on days 14 (2wp1) and 35 (2wp2).

F-specific serum IgG, IgG1, and IgG2c titers (GMT) were as follows:

RNA + liposome RNA + CNE Protein vaccine Day WT Resq WT Resq WT ResqTotal IgG 14 718 401 849 99 2795 2295 35 2786 1650 1978 374 41519 33327IgG 1 14 25 25 136 76 3410 3238 35 125 125 195 183 38150 48040 IgG 2c 141605 849 136 76 25 25 35 14452 3183 7567 335 125 125

These results confirm the previous findings that, unlike the proteinvaccine, the RNA vaccine shows at least a partial dependence on TLR7 forits immunogenicity, particularly with the emulsion adjuvant.

Further Innate Immunity Receptors and Cytokine Responses

As shown above, a delivered replicon can stimulate, wild-type mousedendritic cells to secrete IFN-α and IL-6, but the same response is notseen in dendritic cells from mice which carry the Resq mutation in TLR7.

Similarly, Lipofectamine-delivered vA317 replicons can stimulatewild-type mouse fibroblasts to secrete high levels of IFN-β and IL-6,but the replicons stimulate much lower levels of these cytokines infibroblasts which lack MDA5 or RIG-I i.e., cytoplasmic RNA receptors(see FIGS. 15A and 15B for IFN-β and IL-6 levels respectively). Thesefibroblasts are non-immune cells which do not respond to TLR7 ligands.Mouse embryonic fibroblasts (MEFs) from RIG-I and MDA5 knockout mice(−/−) were stimulated with replicon RNA formulated with lipofectamine2000. Heterozygous littermates (+/−) were used as controls. The RNAstimulates IL-6 and IFN-β in the heterozygous mice but in the knockoutmice the activation is almost completely abrogated. Thus, thesehelicases are important for RNA recognition in non-immune cells.

In general, liposome-delivered RNA replicons were shown to induceseveral serum cytokines within 24 hours of intramuscular injection(IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL-13, MCP-1, and MIP-a),whereas only MIP-1 was induced by naked RNA and liposome alone inducedonly IL-6. IFN-α was shown to contribute to the immune response toliposome-encapsulated RSV-F-encoding replicon because an anti-IFNαreceptor (IFNAR1) antibody reduced F-specific serum IgG a 10-foldreduction after 2 vaccinations.

Expression Kinetics

Experiments on expression kinetics used RNA encoding GFP or the SEAPreporter enzyme. The “vA306” replicon encodes SEAP; the “vA17” repliconencodes GFP; the “vA336” replicon encodes GFP but cannot self-replicate;the “vA336*” replicon is the same as vA336 but was prepared with 10%) ofuridines replaced with 5-methyluridine (M5U); the “vA336**” replicon isthe same as vA336 but 100% of its uridine residues are M5U. BALB/c micewere given bilateral intramuscular vaccinations (50 μL per leg) on day0. Animals, 35 total, were divided into 7 groups (5 animals per group)and were immunized as follows:

-   -   Group 1 Naïve control.    -   Group 2 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in        liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP)        formulated in liposomes.    -   Group 3 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1p g. SEAP) formulated in        liposomes mixed with non-replicating RNA (vA336, 1.0 μg, GFP)        formulated in liposomes.    -   Group 4 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in        liposomes mixed with non-replicating RNA (vA336*, 1.0 μg, GFP)        formulated in liposomes.    -   Group 5 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in        liposomes mixed with non-replicating RNA (vA336**, 1.0 μg, GFP)        formulated in liposomes.    -   Group 6 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in        liposomes mixed with empty liposomes at the same lipid dose as        groups 2-5.    -   Group 7 were given bilateral intramuscular vaccinations (50 μL        per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in        liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP)        formulated in liposomes.

These experiments aimed to see if host responses to RNA might limitprotein expression. Thus, expression was followed for only 6 days,before an adaptive response (antibodies, T cells) would be apparent.Serum SEAP activity (relative light units) at days 0, 3 and 6 were asfollows (GMT):

Day 1 Day3 Day6 1 898 1170 2670 2 1428 4219 28641 3 1702 9250 150472 41555 8005 76043 5 1605 8822 91019 6 10005 14640 93909 7 1757 6248 53497

Replication-competent RNA encoding GFP suppressed the expression of SEAPmore than replication-defective GFP RNA, suggesting a strong hostdefense response against replicating RNA which leads to suppression ofSEAP expression. It is possible that interferons induced in response tothe GFP RNA suppressed the expression of SEAP. Under the hostresponse/suppression model, blocking host recognition of RNA would beexpected to lead to increased SEAP expression, but 5′ methylation of Uresidues in the GFP RNA was not associated with increased SEAP,suggesting that host recognition of RNA was insensitive to 5′methylation.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining, within thescope and spirit of the invention.

Table 1: Useful Phospholipids

DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine

DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate

DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine

DEPE 1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine

DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )

DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine

DLPA 1,2-Dilauroyl-sn-Glycero-3-Phosphate

DLPC 1,2-Dilauroyl-sn-Glycero-3-phosphatidycholine

DLPE 1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine

DLPG 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )

DLPS 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine

DMG 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine

DMPA 1,2-Dimyristoyl-sn-Glycero-3-Phosphate

DMPC 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine

DMPE 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine

DMPG 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )

DMPS 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine

DOPA 1,2-Dioleoyl-sn-Glycero-3-Phosphate

DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine

DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine

DOPG 1,2-Dioleoy 1-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )

DOPS 1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine

DPPA 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate

DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine

DPPE 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine

DPPG 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )

DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine

DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine

DSPA 1,2-Distearoyl-sn-Glycero-3-Phosphate

DSPC 1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine

DSPE 1,2-Distearoyl-sn-Glycero-3-phosphatidylethanolamine

DSPG 1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )]

DSPS 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine

EPC Egg-PC

HEPC Hydrogenated Egg PC

HSPC High purity Hydrogenated Soy PC

HSPC Hydrogenated Soy PC

LYSOPC MYRISTIC 1-Myristoyl-sn-Glycero-3-phosphatidylcholine

LYSOPC PALMITIC 1-Palmitoyl-sn-Glycero-3-phosphatidylcholine

LYSOPC STEARIC 1-Stearoyl-sn-Glycero-3-phosphatidylcholine

Milk Sphingomyelin MPPC 1-Myristoyl,2-palmitoyl-sn-Glycero3-phosphatidylcholine

MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine

PMPC 1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine

POPC 1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylchoine

POPE 1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine

POPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .]

PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine

SMPC 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine

SOPC 1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine

SPPC 1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

REFERENCES

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The invention claimed is:
 1. A method of obtaining a composition, thecomposition comprising lipid particles and messenger ribonucleic acid(mRNA) molecules; the mRNA molecules comprising: (a) a 5′ capnucleoside, (b) a first 5′ ribonucleoside, (c) a triphosphate bridge,and (d) a sequence that encodes a coronavirus spike polypeptideimmunogen; the first 5′ ribonucleoside comprising a 2′-methylatedribose; the 5′ cap nucleoside being linked 5′-to-5′ to the first 5′ribonucleoside by the triphosphate bridge; the lipid particlescomprising lipids comprising: (a) a polyethylene glycol-ylated lipid,(b) cholesterol, (c) an anionic phospholipid or a zwitterionicphospholipid, and (d) a cationic lipid comprising a tertiary amine; andthe lipid particles encapsulating at least half of the mRNA molecules;the method comprising the steps of: (i) mixing the lipids and ethanol,thereby obtaining an ethanolic lipid mixture; (ii) mixing the mRNAmolecules and an aqueous buffer, thereby obtaining an aqueous RNAmixture; (iii) mixing the ethanolic lipid mixture and the aqueous RNAmixture, thereby obtaining an intermediate mixture; and (iv) purifyingthe intermediate mixture, thereby obtaining the composition.
 2. Themethod of claim 1, the mRNA molecules comprising a modified nucleotide.3. The method of claim 2, the 5′ cap nucleoside being a7-methylguanosine.
 4. The method of claim 1, the lipids comprising thezwitterionic phospholipid; and the zwitterionic phospholipid being1,2-distearoyl-sn-glycero-3-phosphocholine.
 5. The method of claim 2,the lipids comprising the zwitterionic phospholipid; and thezwitterionic phospholipid being1,2-distearoyl-sn-glycero-3-phosphocholine.
 6. The method of claim 3,the lipids comprising the zwitterionic phospholipid; and thezwitterionic phospholipid being1,2-distearoyl-sn-glycero-3-phosphocholine.
 7. The method of claim 1,the mRNA molecules being self-replicating RNA.
 8. The method of claim 1,wherein in step (iv), the purifying comprises a tangential flowfiltration.
 9. The method of claim 2, wherein in step (iv), thepurifying comprises a tangential flow filtration.
 10. The method ofclaim 5, wherein in step (iv), the purifying comprises a tangential flowfiltration.
 11. The method of claim 9, wherein in step (iv), thepurifying comprises a tangential flow filtration.
 12. The method ofclaim 1, wherein the ethanolic lipid mixture being heated prior to step(iii).
 13. The method of claim 2, wherein the ethanolic lipid mixturebeing heated prior to step (iii).
 14. The method of claim 5, wherein theethanolic lipid mixture being heated prior to step (iii).
 15. The methodof claim 6, wherein the ethanolic lipid mixture being heated prior tostep (iii).
 16. The method of claim 10, wherein the ethanolic lipidmixture being heated prior to step (iii).
 17. The method of claim 11,wherein the ethanolic lipid mixture being heated prior to step (iii).18. The method of claim 1, wherein the aqueous buffer being a citratebuffer.
 19. The method of claim 2, wherein the aqueous buffer being acitrate buffer.
 20. The method of claim 10, the aqueous buffer being acitrate buffer.
 21. The method of claim 11, wherein the aqueous bufferbeing a citrate buffer.
 22. The method of claim 1, at least 80% of thelipid particles having a diameter in the range of 20-220 nm.
 23. Themethod of claim 2, at least 80% of the lipid particles having a diameterin the range of 20-220 nm.
 24. The method of claim 5, at least 80% ofthe lipid particles having a diameter in the range of 20-220 nm.
 25. Themethod of claim 6, at least 80% of the lipid particles having a diameterin the range of 20-220 nm.
 26. The method of claim 24, wherein in step(iv), the purifying comprises a tangential flow filtration.
 27. Themethod of claim 25, wherein in step (iv), the purifying comprises atangential flow filtration.
 28. The method of claim 8, the tangentialflow filtration comprising a membrane that has a 100 kDa pore sizecutoff.
 29. The method of claim 9, the tangential flow filtrationcomprising a membrane that has a 100 kDa pore size cutoff.
 30. Themethod of claim 10, the tangential flow filtration comprising a membranethat has a 100 kDa pore size cutoff.
 31. The method of claim 11, thetangential flow filtration comprising a membrane that has a 100 kDa poresize cutoff.
 32. The method of claim 20, the tangential flow filtrationcomprising a membrane that has a 100 kDa pore size cutoff.
 33. Themethod of claim 21, the tangential flow filtration comprising a membranethat has a 100 kDa pore size cutoff.
 34. The method of claim 26, thetangential flow filtration comprising a membrane that has a 100 kDa poresize cutoff.
 35. The method of claim 27, the tangential flow filtrationcomprising a membrane that has a 100 kDa pore size cutoff.
 36. Themethod of claim 32, the tangential flow filtration comprising aphosphate-buffered saline.
 37. The method of claim 33, the tangentialflow filtration comprising a phosphate-buffered saline.
 38. The methodof claim 1, the mixing of step (iii) being performed with a T-mixer. 39.The method of claim 2, the mixing of step (iii) being performed with aT-mixer.
 40. The method of claim 3, the mixing of step (iii) beingperformed with a T-mixer.
 41. The method of claim 5, the mixing of step(iii) being performed with a T-mixer.
 42. The method of claim 6, themixing of step (iii) being performed with a T-mixer.
 43. The method ofclaim 10, the mixing of step (iii) being performed with a T-mixer. 44.The method of claim 11, the mixing of step (iii) being performed with aT-mixer.
 45. The method of claim 20, the mixing of step (iii) beingperformed with a T-mixer.
 46. The method of claim 21, the mixing of step(iii) being performed with a T-mixer.